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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction.
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PMID:Effect of phospholipase C on cholesterol solubilization in model bile. A concanavalin A-binding nucleation-promoting factor from human gallbladder bile. 171 7

A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio similar to that found with sn-2[3H]DPPC. Finally, no radiolabeled lysolecithin was identified with [3H]choline-DPPC or [14C]DPPC as substrate indicating the free fatty acid was the product of a diacylglycerol-lipase rather than a phospholipase A2. Phospholipase C and diacyl-glycerol-lipase activities were significantly correlated (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile. 206 34

This study describes the identification of a phospholipase C activity against phosphatidylcholine in delipidated human gallbladder bile. All biles were obtained from cholesterol gallstone patients and were negative on bacterial culture. The biliary enzyme was inhibited by EDTA and had a pH optimum of between 7-8. All of the 15 gallbladders examined contained significant phospholipase C activity (32.85 +/- 8.37 nmol/h/mg delipidated protein). The finding of a phospholipase C in gallbladder bile of patients with cholesterol gallstones may be one of the factors responsible for or related to the rapid in vitro nucleation seen in these biles.
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PMID:Identification of a phosphatidylcholine active phospholipase C in human gallbladder bile. 334 54

Concanavalin A (Con A)-binding glycoproteins accelerate the rate of cholesterol crystal formation as a prelude to gallstone formation. Immunoglobulins (IgM, IgA, and IgG), aminopeptidase N (APN), phospholipase C (pcPLC), and alpha 1-acid glycoprotein from this Con A fraction have all been proposed as candidate promoters. We immunopurified each of the six putative promoters and examined their comparative effects by adding equal amounts to a cholesterol crystal growth assay. The effects of immunoabsorptive removal of each of the specific candidate promoters from native bile were also compared. In additional studies, the potency of these proteins was in the following order: IgM > IgA = AAG > IgG. APN and pcPLC showed no effect on cholesterol crystal growth at their apparent physiological concentrations. In subtractive experiments, only a minor loss (< 10%) of net promoting activity from that of the whole Con A-bound fraction was observed after immunoabsorptive removal of pcPLC, APN, or immunoglobulins. Total removal of AAG, however, showed a far greater loss (/33%) of the net promoting activity. These data indicate that AAG accounts for the greatest portion of net biliary Con A-bound promoting activity derived from currently defined and well-identified glycoproteins. However, more than 60% of total Con A-binding promoting activity remains unaccounted for, indicating the presence of other important and still unidentified promoters in human bile.
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PMID:Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, alpha 1-acid glycoprotein, phospholipase C, and aminopeptidase N1. 810 87

Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. Vesicle growth over 2 h could be detected with phospholipase activity as little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been generated as a result of the initial phospholipase C treatment. The presence of fusion intermediates was confirmed using transmission electron microscopy. In addition, kinetically slow vesicle fusion with intravesicle content mixing and minimal leakage was also confirmed by fluorescence spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50 mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was obtained at 30 min at 25 degrees C with phospholipase C activity. This level of enzyme activity approximates that found in human gallbladder bile (1.2 nmol/min per ml). We conclude that the hydrolysis products of phospholipase C activity can, in very small amounts (3-7 mol% diacylglycerol), lead to destabilization and fusion of cholesterol-saturated biliary vesicles. A reappraisal of the importance of phospholipase C hydrolysis products in the pathogenesis of cholesterol gallstones is warranted based on these observations.
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PMID:Lipid vesicle fusion induced by phospholipase C activity in model bile. 842 56

Bile supersaturation is necessary for cholesterol gallstones to form. Not all people with supersaturated bile form gallstones, however, and additional factors must be present. The role of pronucleating substances has been extensively studied. Of these, proteins, especially mucin, are best understood. Mucin is secreted by the gallbladder epithelium and may act as a nidus for crystal nucleation. Other proteins that may act as pronucleators include alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, phospholipase C, and a small calcium binding protein. The role of antinucleating factors is less well understood. Certain drugs, including octreotide and ceftriaxone, may also predispose to stone formation. Another local factor is gallbladder stasis, a well-known risk factor for pigment stone formation. More recent research has focused on the role of bacterial infection, which has long been believed to be a factor in pigment gallstone formation. Newer data also support a role for infection in cholesterol gallstone pathogenesis. Additionally, genetic factors that may predispose a patient to cholesterol gallstones have been identified in mice and in humans.
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PMID:Gallstone formation. Local factors. 1019 80